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Effects of Positive Pressure in Odontogenic Keratocysts

¹ Department of Oral and Maxillofacial Surgery and ² Department of Dental Anesthesiology, Graduate School of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan;

View larger version (34K): [in a new window]   Figure 1. Effects of positive pressure on the expression of IL-1 and on the plasma membrane permeability. (A) Odontogenic keratocyst epithelial cells were incubated in serum-free DMEM for 15 min at 37°C under atmospheric pressure or 80 mm Hg of positive pressure. Total cellular RNA was extracted, and RT-PCR amplifications were performed at 30 cycles for IL-1, and 27 cycles for ß-actin, as described in MATERIALS & METHODS. (B) Odontogenic keratocyst epithelial cells were cultured in serum-free DMEM for 24 hrs at 37°C under atmospheric pressure or 80 mm Hg of positive pressure. The concentration of IL-1 in the conditioned media was measured by ELISA, as described in MATERIALS & METHODS. Vertical bars indicate mean ± SD (n = 4). *Significant difference between atmospheric pressure and positive pressure at p < 0.05. (C) Odontogenic keratocyst epithelial cells were incubated with 5 µM fluo-3 AM for 30 min at room temperature, as described in MATERIALS & METHODS. The fluorescent intensity for fluo-3 was monitored before (a) and after (b) application of 80 mm Hg positive pressure to the cells. Bar represents 40 µm.

View larger version (15K): [in a new window]   Figure 2. Effects of positive pressure and rhIL-1 on the secretion of MMP-1, MMP-2, MMP-3, and PGE2. (A,B) Odontogenic keratocyst fibroblasts (2 x 104 cells/cm2) (a) and odontogenic keratocyst epithelial cells (4 x 104 cells/cm2) (b) were seeded alone or co-cultured (c), and then incubated in serum-free DMEM for 24 hrs at 37°C under atmospheric pressure or 80 mm Hg of positive pressure. IL-1ra was added 15 min before the application of positive pressure. (C,D) Odontogenic keratocyst fibroblasts (a) and odontogenic keratocyst epithelial cells (b) were incubated in serum-free DMEM for 24 hrs at 37°C in the absence or presence of rhIL-1. (A,C) The culture media (200 µL) were concentrated and subjected to Western immunoblotting for MMP-1 and MMP-3, and the 30-µL culture media were subjected to gelatin zymography. (B,D) The concentration of PGE2 in the culture media was measured by ELISA as described in MATERIALS & METHODS. Vertical bars indicate mean ± SD (n = 4). *Significant difference at p < 0.05.

View larger version (40K): [in a new window]   Figure 3. Effects of rhIL-1 and PGE2 on the expression of RANKL, M-CSF, and OPG mRNAs in odontogenic keratocyst fibroblasts. (A) Effects of rhIL-1 on the expression of RANKL mRNA in odontogenic keratocyst fibroblasts. Odontogenic keratocyst fibroblasts were incubated in serum-free DMEM for 12 hrs at 37°C in the absence or presence of rhIL-1. RT-PCR amplifications were performed at 35 cycles for RANKL and 27 cycles for ß-actin, respectively, as described in MATERIALS & METHODS. (B) Effects of PGE2 on the expression of RANKL, M-CSF, and OPG mRNAs in odontogenic keratocyst fibroblasts. Odontogenic keratocyst fibroblasts were incubated in serum-free DMEM for 12 hrs at 37°C in the absence (lane 1) or presence of 10 nM rhIL-1 (lanes 2 and 3) or 10 µM PGE2 (lane 4). Indomethacin (1 µM) was added 15 min before the application of 10 nM rhIL-1 (lane 3). RT-PCR amplifications were performed at 35 cycles for RANKL, 30 cycles for M-CSF, 25 cycles for OPG, and 27 cycles for ß-actin, respectively, as described in MATERIALS & METHODS.